Cell‐surface exposure of a hybrid 3‐cohesin scaffoldin allowing the functionalization of Escherichia coli envelope

Abstract

Cellulosomes are large plant cell wall degrading complexes secreted by some anaerobic bacteria. They are typically composed of a major scaffolding protein containing multiple receptors called cohesins, which tightly anchor a small complementary module termed dockerin harbored by the cellulosomal enzymes. In the present study, we have successfully cell surface exposed in Escherichia coli a hybrid scaffoldin, Scaf6, fused to the curli protein CsgA, the latter is known to polymerize at the surface of E. coli to form extracellular fibers under stressful environmental conditions. The C-terminal part of the chimera encompasses the hybrid scaffoldin composed of three cohesins from different bacterial origins and a carbohydrate-binding module targeting insoluble cellulose. Using three cellulases hosting the complementary dockerin modules and labeled with different fluorophores, we have shown that the hybrid scaffoldin merged to CsgA is massively exposed at the cell surface of E. coli and that each cohesin module is fully operational.Altogether these data open a new route for a series of biotechnological applications exploiting the cell-surface exposure of CsgA-Scaf6 in various industrial sectors such as vaccines, biocatalysts or bioremediation, simply by grafting the small dockerin module to the desired proteins before incubation with the engineered E. coli.

Progression of Recombinant Cellulolytic and Ethanologenic Escherichia coli for Production of Bioethanol: A Review

“…In this way, cellulase purification become unnecessary. In addition, the cell-surface display of cellulolytic E. coli will increase the surface area to facilitate hydrolytic reactions [20]; [21].…”

Improving the Synthesis Efficiency of Amino Acids Such as L-Lysine by Assembling Artificial Cellulosome Elements Dockerin Protein In Vivo

“…However, when learning the ways to achieve extracellular assembly of cellulosome, things appear to have changed. This structure allows for different enzymes to be assembled by scaffolding proteins in a certain proportion to flexibly regulate the types of enzyme proteins and improve the catalytic efficiency of enzymes [44]. The efficiency of cellulosomes to hydrolyze cellulose is six times that of free enzymes [45][46][47].…”

Conjugated Protein Domains as Engineered Scaffold Proteins

“…The potential utilization of cellulosome hybrids and recombination of its domains for various applications was first acknowledged by Bayer et al, who also generated the first in vitro synthetic cellulosome . Innovations within this field have led to the construction of higher-order cellulosomes, the introduction of noncellulosomal enzymes, and their introduction within organisms, such as bacteria − and S. cerevisae . , The cohesin–dockerin systems have also been utilized for nonmetabolic purposes, such as protein purification, , biosensors, and building blocks for in vitro synthetic biology projects . Early systems used cellulose as the substrate; later, modularity of cohesin-scaffolds and dockerin-fused enzymes was utilized in the field of sustainable biosynthesis. − Recent advances in the field of cellulose degradation have focused on transferring the cellulosomal technology to industrial settings.…”